APTAMER-RNAi THERAPEUTIC  COMPOSITIONS

ABSTRACT

A recombinant nucleic acid comprising an aptamer that binds CD4 and an RNAi sequence that silences the expression of RORγ2 is described herein. Pharmaceutical compositions comprising the recombinant nucleic acid, particularly topical compositions are also described. Methods of treating inflammatory disease using the pharmaceutical composition are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/862,065, filed Sep. 22, 2015, which claims the benefit of U.S.Provisional Patent Application No. 62/054,135, filed Sep. 23, 2014, theentire contents of each of which are incorporated herein in theirentirety.

FIELD

Generally, the field is nucleic acid compositions used in the treatmentof disease. More specifically, the field is nucleic acid compositionscomprising an aptamer that binds CD4 fused to an RNAi molecule thatsilences RORγt.

BACKGROUND

RNA interfering (RNAi)-mediated gene silencing holds great promise formanipulating T cells to study basic T cell biology and for developingpotential T cell targeted therapeutics.

Many autoimmune and/or inflammatory diseases are mediated by Th17 cells.These include rheumatoid arthritis, multiple sclerosis, systemic lupuserythematosus, psoriasis, inflammatory bowel disease, and type 1diabetes mellitus (Rao D D et al, Adv Drug Deliv Rev 61, 746-759 (2009);incorporated by reference herein.

The retinoic acid receptor related orphan receptor gamma 2 gene encodesRORγt. RORγt is required for the differentiation of Th17 cells. Th17cells produce several inflammatory cytokines, including IL-17, IL-17F,IL-22 and IL-21 which all have been implicated in mediating the chronicinflammation that is characteristic of a number of autoimmuneinflammatory diseases.

New therapeutics that specifically target Th17 cells rather thantargeting members of the IL-17 family or ubiquitous IL-17 receptors areneeded.

SUMMARY

Disclosed herein are recombinant polyribonucleotides comprising a firstsequence that includes an aptamer that specifically binds CD4 and asecond sequence that includes an RNAi that silences RORγt. Thepolynucleotide can be chemically synthesized or transcribed from a DNAtemplate (including in vitro transcribed.) In some examples, thepolyribonucleotide includes a CD4-specific aptamer sequence encoded bySEQ ID NO: 5. In other examples, the polyribonucleotide includes a RORγtspecific RNAi sequence encoded by SEQ ID NO: 7. The polyribonucleotidecan include a 2′-fluroribonucleic acid. One example of the recombinantpolyribonucleotide is a polyribonucleotide that includes SEQ ID NO: 1.

Disclosed herein are expression vectors comprising the recombinantpolyribonucleotides described herein operably linked to a promoter. Insome examples, the expression vector is stably transfected in a cell.

Disclosed herein are pharmaceutical compositions comprising an effectiveamount of the recombinant polyribonucleotides described herein,including pharmaceutical compositions formulated for topicaladministration.

Disclosed herein are methods of treating diseases mediated by Th17 cellsin subject. Those methods include administering the pharmaceuticalcompositions described herein to the subject. Such diseases includearthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis,inflammatory bowel disease, and type 1 diabetes mellitus.

Disclosed herein are pharmaceutical compositions for use in treatingdiseases mediated by Th17 cells in a subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A is an image of a gel showing the indicated chimeras in vitrotranscribed by T7 RNA polymerase analyzed by denatured PAGE andvisualized by ethidium bromide staining. Lane 1, ssRNA ladder; Lane 2,CD4-AshR-RORγt chimera (SEQ ID NO: 1); Lane 3, mock-CD4-AshR-RORγtchimera (SEQ ID NO: 2).

FIG. 1B is a diagram of the predicted secondary structure ofCD4-AshR-RORγt chimera of SEQ ID NO: 1. The CD4 aptamer (clone 9 inDavis K A et al, Nuc Acids Res 26, 3915-39242 (1998); incorporated byreference herein) responsible for binding to CD4 is outlined. The shRNAportion of the chimera consists of targeted RORγt RNAi with 2 overhangnucleotides at its 3′ end and a 7 nucleotide loop.

FIG. 1C is a diagram of the mock-CD4-AshR-RORγt chimera of SEQ ID NO: 2.

FIG. 1D is an image of a gel resulting from a cleavage analysis ofsynthesized chimeras by Dicer. Lane 1, ssRNA ladder; Lane 2, antisenseRNAi to RORγt; Lane 3, intact CD4-AshR-RORγt chimera; Lanes 4-6 includechimeras that were digested with Dicer: Lane 4, Mock CD4-AshR-RORγtchimera (SEQ ID NO: 2); Lane 5, CD4-AshR-ROyt chimera (SEQ ID NO: 1);Lane 6, CD4-AshR-scrambled control chimera SEQ ID NO: 3.

FIG. 2A is an image of two gels showing CD4- and mock-CD4-AshR-RORγtchimeras labeled by incorporating Cy3-CTP during in vitro transcription.Cy3 scanning (right panel) showed strong Cy3-signaling bands that wereat an appropriate size of transcripts shown in ethidium bromide imaging(left panel). Lane 1, ssRNA ladder; Lane 2, CD4-AshR-RORγt chimera; Lane3, mock-CD4-AshR-RORγt chimera.

FIG. 2B is a set of three images showing uptake of Cy3-labeledCD4-AshR-RORγt chimera by the CD4⁺ human T cell line Karpas 299.

FIG. 2C is a set of six images showing uptake of Cy3 labeledCD4-AshR-RORγt chimera into CD4⁺ T cells in PBMCs (left panels). Note nouptake of Cy3 labeled mock CD4-AshR-RORγt chimera in the right panels.

FIG. 2D is a set of three flow cytometry plots showing that Cy3-labeledCD4-AshR-RORγt chimera was significantly internalized in CD4+ Karpas 299cells and CD4+ T cells but not in CD8⁺ T cells. There is no uptake ofCy3-labeled mock-CD4-AshR-RORγt chimera by Karpas 299 cells or T-cellenriched PBMCs. Gray: PBS; Red line: Cy3-labeled CD4-AshR-RORγt chimera;Blue line: Cy3-labeled mock-CD4-AshR-RORγt chimera (representative of2-5 experiments)

FIG. 3A is a bar graph depicting the results of a quantitative real-timePCR assay for RORγt gene expression. RORγt gene expression wassignificantly reduced by CD4-AshR-RORγt chimera in aconcentration-dependent manner in Karpas 299 cells.

FIG. 3B is a bar graph depicting the results of a quantitative real-timePCR assay for RORγt gene expression in T-cell enriched PBMCsMock-CD4-AshR-RORγt chimera, CD4-AshR-scrambled control or CD4-AshR-CCR5chimera had no effect on RORγt gene expression in T-cell enriched PBMCs.

FIG. 3C is a set of three bar graphs showing that all the chimeraslacked a significant inhibition on TBX21 or GATA3 in T-cell enrichedPBMCs. (Data are presented as mean±SD of three experiments). 1, PBS; 2,CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4,CD4-AshR-scrambled control chimera; 5, CD4-AshR-CCR5 chimera.

FIG. 3D is a FACS plot showing RORγt protein expression analyzed by flowcytometry in Karpas 299 cells were stimulated with PMA 50 ng/ml for 24h. Red line, CD4-AshR-RORγt chimera; Blue line, Mock-CD4-AshR-RORγtchimera (representative of three experiments). The x-axis indicatesRORγt expression with higher expression on the right.

FIG. 3E is a FACS plot showing RORγt protein expression in PBMCsstimulated with anti-CD3/CD28 and LPS for 48 h. RORγt expression wasreduced by CD4-AshR-RORγt chimera (red line), but not bymock-CD4-AshR-RORγt chimeras (blue line) or CD4-AshR-scrambled controlchimera (purple line). Black line, PBMCs without stimulation; greenline, PBMCs with stimulation but without chimeras (representative ofthree experiments). The x-axis indicates RORγt expression with higherexpression on the right.

FIG. 3F is a bar graph showing the percentage of RORγt⁺ cells instimulated T-cell enriched PBMCs was reduced by CD4-AshR-RORγt, but notby mock-CD4-AshR-RORγt chimera or CD4-AshR-scrambled control chimera. 1,PBMCs without stimulation; 2, PBMCs with stimulation but withoutchimeras; 3, Stimulated PBMCs were treated with CD4-AshR-RORγt chimeras;4, Stimulated PBMCs were treated with mock-CD4-AshR-RORγt chimera; 5,Stimulated PBMCs were treated with CD4-AshR-scrambled control chimera(Data are presented as mean±SD of three experiments).

FIG. 4A is a bar graph showing expression of IL-17A in the supernatantof Karpas 299 cells stimulated with PMA measured by ELISA. X-axis showsconcentration of added CD4-AshR-RORγt.

FIG. 4B is a bar graph showing expression of IL-17A in the supernatantof T cell enriched PBMC stimulated with anti-CD3/CD28 measured by ELISA.X-axis shows concentration of CD4-AshR-RORγt.

FIG. 4C is a FACS plot showing fewer IL-17A producing CD4⁺ T cells inthe presence of CD4-AshR-RORγt chimera relative to negative controls.

FIG. 4D is a bar graph recapitulating the data from FIG. 4C. 1, PBS; 2;CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4,CD4-AshR-scrambled control chimera (Data are presented as mean±SD ofthree experiments).

FIG. 4E is a FACS plot showing no effect of IFN-y producing CD4⁺ T cellsin PBMC in the presence of CD4-AshR-RORγt chimera relative to negativecontrols.

FIG. 4F is a bar graph recapitulating the data from FIG. 4E. 1, PBS; 2;CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4,CD4-AshR-scrambled control chimera.

FIG. 4G is a FACS plot showing no effect of IL-4 producing CD4⁺ T cellsin PBMC in the presence of CD4-AshR-RORγt chimera relative to negativecontrols.

FIG. 4H is a bar graph recapitulating the data from FIG. 4E. 1, PBS; 2;CD4-AshR-RORγt chimera; 3, mock-CD4-AshR-RORγt chimera; 4,CD4-AshR-scrambled control chimera.

SEQUENCE LISTING

SEQ ID NO: 1 is a CD4-aptamer RORγt shRNA chimera.

SEQ ID NO: 2 is a negative control chimera that includes a mock CD4binding aptamer and an intact RORγt shRNA.

SEQ ID NO: 3 is a negative control chimera that includes an intact CD4binding aptamer and a scrambled shRNA

SEQ ID NO: 4 is a negative control chimera that includes an intact CD4binding aptamer and a CCR5 shRNA.

SEQ ID NO: 5 is a DNA sequence encoding a CD4 binding aptamer. SEQ IDNO: 6 is a DNA sequence encoding a negative control aptamer.

SEQ ID NO: 7 is a DNA sequence encoding a RORγt shRNA.

SEQ ID NO: 8 is a DNA sequence encoding a scrambled shRNA.

SEQ ID NO: 9 is a DNA sequence encoding a CCR5 shRNA.

DETAILED DESCRIPTION Terms

Unless otherwise noted, technical terms are used according toconventional usage.

Definitions of common terms in molecular biology may be found inBenjamin Lewin, Genes V, published by Oxford University Press, 1994(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia ofMolecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCRPublishers, Inc., 1995 (ISBN 1-56081-569-8)

In order to facilitate review of the various embodiments of thedisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as atherapeutic agent, by any effective route. Exemplary routes ofadministration include, but are not limited to, injection (such assubcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), oral, intraductal, sublingual, rectal, transdermal,topical, intranasal, vaginal and inhalation routes.

Aptamer: An oligonucleotide of a single defined sequence that bindsspecifically to a target molecule. An aptamer can be single- ordouble-stranded. In some examples an aptamer is conjugated to an RNAiand can thereby deliver the RNAi to a cell expressing a target molecule(such as a protein) to which the aptamer can specifically bind. Aptamerscan range between 10 and 100 nucleotides in length, including 15 to 40nucleotides or 20 to 40 nucleotides.

Binding or stable binding: An association between two substances ormolecules, such as the association of an aptamer with a protein or othertarget molecule. Binding can be detected by any procedure known to oneskilled in the art, such as by physical or functional properties.Binding can be determined by binding a labeled version of an aptamer tothe target molecule, then detecting the presence of the target on cellsalso known to express the target molecule using, for example, two colorflow cytometry. Binding can be detected functionally by determiningwhether binding has an observable effect upon a biosynthetic processsuch as expression of a gene (including RNAi mediated silencing), DNAreplication, transcription, translation, protein activity and the like.

Contacting: Placement in direct physical association, includingcontacting of a solid with a solid, a liquid with a liquid, a liquidwith a solid, or either a liquid or a solid with a cell or tissue,whether in vitro or in vivo. Contacting can occur in vitro with isolatedcells or tissue or in vivo by administering to a subject.

CD4: (Cluster of differentiation 4) is a glycoprotein found on thesurface of various cells of the immune system. CD4 is expressed mostpredominantly on helper T cells (Th) cells, but it may also be expressedby antigen presenting cells such as macrophages and dendritic cells.

Effective amount: An amount of agent, such as a polyribonucleotidecomprising SEQ ID NO: 1 that is sufficient to generate a desiredresponse, such as the reduction or elimination of a sign or symptom of acondition or disease, such as a Th17 mediated disease. Alternatively, aneffective amount may be an amount sufficient to generate a desiredresponse in an animal model of a Th17 mediated disease.

When administered to a subject, a dosage will generally be used thatwill achieve target tissue concentrations that have been shown toachieve activity in vitro. In some examples, an “effective amount” isone that prophylactically treats one or more symptoms and/or underlyingcauses of a disorder or disease. An effective amount can also be anamount that therapeutically treats one or more symptoms and/orunderlying causes of a disorder or disease.

Interfering RNA: Interfering RNA (which ca n be interchangeably referredto as RNAi) or an interfering RNA sequence refers to double-stranded RNAthat is capable of silencing, reducing, or inhibiting expression of atarget gene by any mechanism of action now known or yet to be disclosed.For example, RNAi may act by mediating the degradation of mRNAs whichare complementary to the sequence of the RNAi when the RNAi is in thesame cell as the target gene. RNAi thus refers to both thedouble-stranded RNA formed by two complementary RNA strands or by asingle, self-complementary strand. RNAi may have substantial or completeidentity to the target gene or may comprise one or more mismatches uponalignment to the target gene. The sequence of the interfering RNA maycorrespond to the full length target gene, or any subsequence thereof.As described herein an RNAi is an interfering RNA sequence that ischemically synthesized as an oligoribonucleotide. An RNAi can be singlestranded or double stranded. As described herein an shRNA is an RNAimolecule that is expressed from a DNA template using a promoter operablylinked to the shRNA sequence. The shRNA ca n be expressed in vitro orexpressed upon transfection of a cell with an expression vectorcomprising the shRNA and a cell specific promoter. A shRNA includes anRNAi and its complementary strand with a linker sequence between. TheshRNA is transcribed as a single RNA molecule that forms a doublestranded structure after transcription.

Nucleic acid synthesis and Purification: Methods for isolating RNA,synthesizing RNA, hybridizing nucleic acids, making and screening cDNAlibraries, and performing PCR are well known in the art (see, e.g.,Gubler and Hoffman, Gene 25, 263-269 (1983); Sambrook and Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor N.Y., (2001)) as are PCR methods (see, U.S.Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods andApplications, Innis et al, eds, (1990)). Expression libraries are alsowell known to those of skill in the art. Additional basic textsdisclosing the general methods of use in this invention include Sambrookand Russell (2001) supra; Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994).

Operably Linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in such a way that it has an effect upon the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Operably linked DNA sequences may be contiguous, orthey may operate at a distance.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E.W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the compositions disclosed herein. In general, the nature ofthe carrier will depend on the particular mode of administration beingemployed. For instance, formulations intended for topical use includepharmaceutically and physiologically acceptable fluids such as water,physiological saline, balanced salt solutions, aqueous dextrose,glycerol or the like as a vehicle as well as other compounds thatpromote penetration into the skin.

In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered ca n contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Promoter: A promoter may be any of a number of nucleic acid controlsequences that direct transcription of a nucleic acid. Typically, aeukaryotic promoter includes necessary nucleic acid sequences near thestart site of transcription, such as, in the case of a polymerase IItype promoter, a TATA element or any other specific DNA sequence that isrecognized by one or more transcription factors. Expression by apromoter can be further modulated by enhancer or repressor elements.Numerous examples of promoters are available and well known to those ofskill in the art. A nucleic acid comprising a promoter operably linkedto a nucleic acid sequence that codes for a particular polypeptide ca nbe termed an expression vector.

Quantification of gene expression: Methods known in the art for thedetection and quantification of RNA expression in a sample includenorthern blotting and in situ hybridization (Parker and Barnes, Methodsin Molecular Biology 106, 247-283 (1999); RNAse protection assays (Hod,Biotechniques 13, 852-854 (1992)); and PCR-based methods, such asreverse transcription polymerase chain reaction (RT-PCR) (Weis et al.,Trends in Genetics 8, 263-264 (1992)). Representative methods forsequencing-based gene expression analysis include Serial Analysis ofGene Expression (SAGE), and gene expression analysis by massivelyparallel signature sequencing (MPSS). (See Mardis E R, Annu Rev GenomicsHum Genet 9, 387-402 (2008)).

For example: proteins can be detected and quantified through epitopesrecognized by polyclonal and/or monoclonal antibodies used in methodssuch as ELISA, immunoblot assays, flow cytometric assays,immunohistochemical assays, radioimmunoassays, Western blot assays, animmunofluorescent assays, chemiluminescent assays and other polypeptidedetection strategies. Proteins may also be detected by mass spectrometryassays (potentially coupled to immunoaffinity assays) includingmatrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)mass mapping and liquid chromatography/quadrupole time-of-flightelectrospray ionization tandem mass spectrometry (LC/Q-TOF-ESI-MS/MS).Additionally, protein expression may be detected by tagging of proteinsseparated by two-dimensional polyacrylamide gel electrophoresis(2D-PAGE), (Kiernan et al, Anal Biochem 301, 49-56 (2002); Poutanen etal, Mass Spectrom 15, 1685-1692 (2001)) or any other method of detectingprotein.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two or more otherwise separated segments ofsequence. This artificial combination is often accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mal.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Carpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mal. Bio. 24:307-31, 1994. Altschul et al., J.Mal. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mal. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation ca n be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. If the two compared sequences sharehomology, then the designated output file will present those regions ofhomology as aligned sequences. If the two compared sequences do notshare homology, then the designated output file will not present alignedsequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences.

The percent sequence identity is determined by dividing the number ofmatches either by the length of the sequence set forth in the identifiedsequence, or by an articulated length (such as 100 consecutivenucleotides or amino acid residues from a sequence set forth in anidentified sequence), followed by multiplying the resulting value by100. For example, a nucleic acid sequence that has 1166 matches whenaligned with a test sequence having 1154 nucleotides is 75.0 percentidentical to the test sequence (1166÷1554*100=75.0). The percentsequence identity value is rounded to the nearest tenth. For example,75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15,75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length valuewill always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(that is, 15÷20*100=75).

For comparisons of amino 5 acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost 5 of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs use SEG. In addition, a manual alignment can beperformed. Proteins with even greater similarity will show increasingpercentage identities when assessed by this method, such as at leastabout 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to aprotein.

When aligning short peptides (fewer than around 30 amino acids), thealignment is performed using the Blast 2 sequences function, employingthe PAM30 matrix set to default parameters (open gap 9, extension gap 1penalties). Proteins with even greater similarity to the referencesequence will show increasing percentage identities when assessed bythis method, such as at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% sequence identity to a protein. When less than the entiresequence is being compared for sequence identity, homologs willtypically possess at least 75% sequence identity over short windows of10-20 amino acids, and ca n possess sequence identities of at least 85%,90%, 95% or 98% depending on their identity to the reference sequence.Methods for determining sequence identity over such short windows aredescribed at the NCBI web site.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. An alternative (and not necessarilycumulative) indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

One of skill in the art will appreciate that the particular sequenceidentity ranges are provided for guidance only; it is possible thatstrongly significant homologs could be obtained that fall outside theranges provided.

Severe combined immunodeficiency (SCID) mouse: Refers to a strain ofmice that is unable to undergo V(D)J recombination and therefore lackfunctional T cells and B cells. SCID mice also have an impaired abilityto activate some components of the complement system. SCID mice arehomozygous for the Prkdc^(scid) mutation.

Subject: A living multicellular vertebrate organism, a category thatincludes, for example, mammals and birds. A mammal includes both humanand non-human mammals, such as mice or non-human primates. In someexamples, a subject is a patient, such as a patient that displays signsor symptoms of psoriasis. In other examples a subject is an experimentalsubject such as an immunocompromised mouse that has been grafted withhuman psoriatic skin tissue.

T cell: A type of lymphocyte (a subset of white blood cells) that playsa central role in cell-mediated immunity. T cells are distinguished fromother types of lymphocytes, such as B cells and NK cells, by thepresence of a special receptor on their cell surface that is called theT cell receptor (TCR). The thymus is generally believed to be theprincipal organ for T cell development. There are many different typesof T cells and these types can be differentiated by the type of T cellreceptor that they express (α/β or γ/δ) by the expression of particularmarkers (CD4⁺, CD8⁺, etc.), by their function (helper Tcells—abbreviated as Th, cytotoxic T cells—abbreviated as Tc or CTL,etc.) T cells of a particular group can be subtyped. For example, helperT cells can be differentiated further into Th1, Th2, Th17, and othercell types based on the types of factors expressed by the T cell as wellas the function of the T cell.

Treating a disease: Inhibiting the full development of a disease orcondition, for example, in a subject who has or is at risk fordeveloping a disease such as psoriasis. Treatment refers to anytherapeutic intervention that ameliorates a sign or symptom of a diseaseor pathological condition. The term “ameliorating,” with reference to adisease or pathological condition, refers to any observable beneficialeffect provided by a pharmaceutical composition. The beneficial effectcan be evidenced, for example, by a delayed onset of clinical symptomsof the disease in a susceptible subject, a reduction in severity of someor all clinical symptoms of the disease, a slower progression of thedisease or by other clinical or physiological parameters associated witha particular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

Vector: A nucleic acid molecule allowing insertion of foreign nucleicacid without disrupting the ability of the vector to replicate and/orintegrate in a host cell. A vector can include nucleic acid sequencesthat permit it to replicate in a host cell, such as an origin ofreplication. A vector ca n also include one or more selectable markergenes and other genetic elements. An integrating vector is capable ofintegrating itself into a host nucleic acid. An expression vector is avector that contains the necessary regulatory sequences to allowtranscription and translation of inserted gene or genes. In one exampledescribed herein, the vector comprises a nucleic acid sequence thatencodes CD4-AsiC-ROR2.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below.

RNAi Based Therapeutics

The concept of RNAi includes small-interfering RNA, which mayinterchangeably be referred to as RNAi and small hairpin RNA referred toas shRNA. An RNAi construct may be any interfering RNA with a duplexlength of about 15-60, 15-50, or 15-40 nucleotides in length, moretypically about 15-30, 15-25, or 18-23 nucleotides in length. Eachcomplementary sequence of the double-stranded RNAi may be 15-60, 15-50,15-40, 15-30, 15-25, or 18-23 nucleotides in length, but othernoncomplementary sequences may be present. For example, RNAi duplexesmay comprise 3′ overhangs of 1 to 4 or more nucleotides and/or 5′phosphate termini comprising 1 to 4 or more nucleotides. An RNAi may besynthesized in any of a number of conformations. One skilled in the artwould recognize the type of RNAi conformation to be used for aparticular purpose. Examples of RNAi conformations include, but need notbe limited to, a double-stranded polynucleotide molecule assembled fromtwo separate stranded molecules, wherein one strand is the sense strandand the other is the complementary antisense strand; a double-strandedpolynucleotide molecule assembled from a single-stranded molecule, wherethe sense and antisense regions are linked by a nucleic acid-based ornon-nucleic acid-based linker; a double-stranded polynucleotide moleculewith a hairpin secondary structure having complementary sense andantisense regions; or a circular single-stranded polynucleotide moleculewith two or more loop structures and a stem having self-complementarysense and antisense regions. In the case of the circular polynucleotide,the polynucleotide may be processed either in vivo or in vitro togenerate an active double-stranded RNAi molecule.

An RNAi can be chemically synthesized, may be encoded by a plasmid andtranscribed, or may be vectored by a virus engineered to express thesiRNA. A RNAi may be a single stranded molecule with complementarysequences that self-hybridize into duplexes with

15 hairpin loops. RNAi can also be generated by cleavage of parent dsRNAthrough the use of an appropriate enzyme such as E. coli RNase III orDicer (Yang et al, Proc. Natl. Acad. Sci. USA 99, 9942-9947 (2002);Calegari et al, Proc. Natl. Acad. Sci. USA 99, 14236-14240 (2002); Byromet al, Ambion TechNotes 10, 4-6 (2003); Kawasaki et al, Nucleic AcidsRes 31, 981-987 (2003); Knight et al, Science 293, 2269-2271 (2001); andRobertson et al, J Biol Chem 243, 82-91 (1968)). A parent dsRNA may beany double stranded RNA duplex from which an RNAi may be produced, suchas a full or partial mRNA transcript.

A mismatch motif may be any portion of a RNAi sequence that is not 100%complementary to its target sequence. A RNAi may have zero, one, two, orthree or more mismatch regions. The mismatch regions may be contiguousor may be separated by any number of complementary nucleotides. Themismatch motifs or regions may comprise a single nucleotide or maycomprise two or more consecutive nucleotides.

A RNAi molecule ca n inhibit the expression of a target gene, such as agene involved in the development of psoriasis such as RORC2. The terms“silencing” or “reducing” may be used interchangeably with “inhibiting.”To examine the extent of inhibition of expression by a siRNA, a RNAi ofinterest is added to a test sample and to a negative control sample towhich the RNAi was not added. Preferably, a negative control sample issimilar to the test sample. More preferably, the negative control sampleis identical to the test sample. Examples of negative control samplesinclude untreated samples, samples to which a siRNA-free buffer wasadded, or samples to which a negative control or mock RNAi was added.Expression in the test sample is then compared to expression in thenegative control sample. Expression may be measured by the detection ofany expression product known in the art or yet to be disclosed. Typicalexpression products that may be detected include RNA or protein.

RNAi molecules can be provided in several forms including, e.g., as oneor more isolated RNAi duplexes, as longer double-stranded RNA (dsRNA),or as RNAi or dsRNA transcribed from a transcriptional cassette in a DNAplasmid. The RNAi sequences may have overhangs (as 3′ or 5′ overhangs asdescribed in Elbashir et al, Genes Dev 15, 188 (2001) or Nykanen et al,Cell 107, 309 (2001)) or may lack overhangs (i.e., have blunt ends).

Expression vectors encoding one or more RNAi templates may be used toprovide siRNA.

RNAi ca n be transcribed as sequences that automatically fold intoduplexes with hairpin loops from DNA templates in plasmids having RNApolymerase Ill transcriptional units, for example, based on thenaturally occurring transcription units for small nuclear RNA U6 orhuman RNase P RNA HI (Brummelkamp et al, Science 296, 550 (2002); Donzeet al, Nucleic Acids Res 30, e46 (2002); Paddison et al, Genes Dev 16,948 (2002); Yu et al, Proc Natl Acad Sci USA 99, 6047 (2002); Lee et al,Nat Biotech, 20, 500 (2002); Miyagishi et al, Nat Biotech 20, 497(2002); Paul et al, Nat Biotech, 20, 505 (2002); and Sui et al, ProcNatl Acad Sci USA, 99, 5515 (2002)).

Typically, a transcriptional unit or cassette will contain an RNAtranscript promoter sequence, such as an H1-RNA or a U6 promoter,operably linked to a template for transcription of a desired RNAisequence and a termination sequence, comprised of 2-3 uridine residuesand a polythymidine (TS) sequence (polyadenylation signal) (Brummelkampet al (2002) supra). The selected promoter can provide for constitutiveor inducible transcription. Compositions and methods for DNA-directedtranscription of RNA interference molecules are described in detail inU.S. Pat. No. 6,573,099. The transcriptional unit is incorporated into aplasmid or DNA vector from which the interfering RNA is transcribed.Plasmids suitable for in vivo delivery of genetic material fortherapeutic purposes are described in detail in U.S. Pat. Nos. 5,962,428and 5,910,488. The selected plasmid ca n provide for transient or stabledelivery of a nucleic acid to a target cell. It will be apparent tothose of skill in the art that plasmids originally designed to expressdesired gene sequences can be modified to contain a transcriptional unitcassette for transcription of siRNA.

A RNAi molecule may be chemically synthesized. In one example ofchemical synthesis, a single-stranded nucleic acid that includes theRNAi duplex sequence can be synthesized using any of a variety oftechniques known in the art, such as those described in Usman et al, JAm Chem Soc, 109, 7845 (1987); Scaringe et al, Nucl Acids Res, 18, 5433(1990); Wincott et al, Nucl Acids Res, 23, 2677-2684 (1995); and Wincottet al, Methods Mol Bio 74, 59 (1997). Synthesis of the single-strandednucleic acid makes use of common nucleic acid protecting and couplinggroups, such as dimethoxytrityl at the 5′-end and phosphoramidites atthe 3′-end. As a non-limiting example, small scale syntheses ca n beconducted on an Applied Biosystems synthesizer using a 0.2 micromolarscale protocol with a 2.5 min coupling step for 2′-0-methylatednucleotides. Alternatively, syntheses at the 0.2 micromolar scale ca nbe performed on a 96-well plate synthesizer from Protogene. However, alarger or smaller scale of synthesis is encompassed by the invention,including any method of synthesis now known or yet to be disclosed.Suitable reagents for synthesis of the RNAi single-stranded molecules,methods for RNA deprotection, and methods for RNA purification are knownto those of skill in the art.

An RNAi can also be synthesized via a tandem synthesis technique,wherein both strands are synthesized as a single continuous fragment orstrand separated by a linker that is subsequently cleaved to provideseparate fragments or strands that hybridize to form an RNAi duplex. Thelinker may be any linker, including a polynucleotide linker or anon-nucleotide linker. The tandem synthesis of RNAi can be readilyadapted to both multiwell/multiplate synthesis platforms as well aslarge scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, the RNAi ca n be assembled fromtwo distinct single-stranded molecules, wherein one strand includes thesense strand and the other includes the antisense strand of the siRNA.For example, each strand can be synthesized separately and joinedtogether by hybridization or ligation following synthesis and/ordeprotection. Either the sense or the antisense strand may containadditional nucleotides that are not complementary to one another and donot form a double stranded siRNA. In certain other instances, the RNAimolecules can be synthesized as a single continuous fragment, where theself-complementary sense and antisense regions hybridize to form a RNAiduplex having hairpin secondary structure.

An RNAi molecule may comprise a duplex having two complementary strandsthat form a double-stranded region with least one modified nucleotide inthe double-stranded region. The modified nucleotide may be on one strandor both. If the modified nucleotide is present on both strands, it maybe in the same or different positions on each strand. A modified RNAimay be less immunostimulatory than a corresponding unmodified RNAisequence, but retains the capability of silencing the expression of atarget sequence.

Examples of modified nucleotides suitable for use in the presentinvention include, but are not limited to, ribonucleotides having a2′-0-methyl (2′0Me), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-0-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a conformation such as those described inthe art, for example in Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in RNAi molecules.Other modified nucleotides include, without limitation: locked nucleicacid (LNA) nucleotides, G-clamp nucleotides, or nucleotide base analogs.LNA nucleotides include but need not be limited to 2′-0,4′-C-methylene-(D-ribofuranosyl)nucleotides), 2′-0-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2Cl) nucleotides, and 2′-azidonucleotides. A G-clamp nucleotide refers to a modified cytosine analogwherein the modifications confer the ability to hydrogen bond bothWatson-Crick and Hoogsteen faces of a complementary guanine nucleotidewithin a duplex (Lin et al, J Am Chem Soc, 120, 8531-8532 (1998)).Nucleotide base analogs include for example, C-phenyl, C-naphthyl, otheraromatic derivatives, inosine, azole carboxamides, and nitroazolederivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and6-nitroindole (Loakes, Nucl Acids Res, 29, 2437-2447 (2001)).

A RNAi molecule may comprise one or more chemical modifications such asterminal cap moieties, phosphate backbone modifications, and the like.Examples of classes of terminal cap moieties include, withoutlimitation, inverted deoxy abasic residues, glyceryl modifications,4′,5′-methylene nucleotides, 1-β-D-erythrofuranosyl) nucleotides,4′-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitolnucleotides, L-nucleotides, a-nucleotides, modified base nucleotides,threo pentofuranosyl nucleotides, acyclic 3′,4′-seco nucleotides,acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentylnucleotides, 3′-3′-inverted nucleotide moieties, 3′-3′-inverted abasicmoieties, 3′-2′-inverted nucleotide moieties, 3′-2′-inverted abasicmoieties, 5′-5′-inverted nucleotide moieties, 5′-5′-inverted abasicmoieties, 3′-5′-inverted deoxy abasic moieties, 5′-amino-alkylphosphate, 1,3-diamino-2-propyl phosphate, 3 aminopropyl phosphate,6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropylphosphate, 1,4-butanediol phosphate, 3′-phosphoramidate, 5′phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al, Tetrahedron 49,1925 (1993)). Non-limiting examples of phosphate backbone modifications(i.e., resulting in modified internucleotide linkages) includephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al,Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al,Antisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA.

The sense and/or antisense strand of an RNAi may comprise a 3′-terminaloverhang having 1 to 4 or more 2′-deoxyribonucleotides and/or anycombination of modified and unmodified nucleotides. Additional examplesof modified nucleotides and types of chemical modifications that can beintroduced into the modified RNAi molecules of the present invention aredescribed, e.g., in UK Patent No. GB 2,397,818 B and U.S. PatentPublication Nos. 20040192626 and 20050282188.

An RNAi molecule may comprise one or more non-nucleotides in one or bothstrands of the siRNA. A non-nucleotide may be any subunit, functionalgroup, or other molecular entity capable of being incorporated into anucleic acid chain in the place of one or more nucleotide units that isnot or does not comprise a commonly recognized nucleotide base such asadenosine, guanine, cytosine, uracil, or thymine, such as a sugar orphosphate.

Chemical modification of the RNAi may also comprise attaching aconjugate to the RNAi molecule. The conjugate can be attached at the 5′-and/or the 3′-end of the sense and/or the antisense strand of the RNAivia a covalent attachment such as a nucleic acid or non-nucleic acidlinker. The conjugate can also be attached to the RNAi through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). A conjugatemay be added to the RNAi for any of a number of purposes. For example,the conjugate may be a molecular entity that facilitates the delivery ofthe RNAi into a cell or the conjugate a molecule that comprises a drugor label. Examples of conjugate molecules suitable for attachment to theRNAi of the present invention include, without limitation, steroids suchas cholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Other examples include the2′-0-alkyl amine, 2′-0-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples of conjugate moleculesinclude a hydrophobic group, a membrane active compound, a cellpenetrating compound, a cell targeting signal, an interaction modifier,or a steric stabilizer as described in U.S. Patent Publication No.20040167090. Further examples include the conjugate molecules describedin U.S. Patent Publication No. 20050239739.

The type of conjugate used and the extent of conjugation to the RNAi canbe evaluated for improved pharmacokinetic profiles, bioavailability,and/or stability of the RNAi while retaining activity. As such, oneskilled in the art can screen RNAi molecules having various conjugatesattached thereto to identify RNAi conjugates having improved propertiesusing any of a variety of well-known in vitro cell culture or in vivoanimal models.

An RNAi may be incorporated into a carrier systems containing the RNAimolecules described herein. The carrier system may be a lipid-basedcarrier system such as a stabilized nucleic acid-lipid particle (e.g.,SNALP or SPLP), cationic lipid or liposome nucleic acid complexes (i.e.,lipoplexes), a liposome, a micelle, a virosome, or a mixture thereof. Inother embodiments, the carrier system is a polymer-based carrier systemsuch as a cationic polymer-nucleic acid complex (i.e., polyplex). Inadditional embodiments, the carrier system is a cyclodextrin-basedcarrier system such as a cyclodextrin polymer-nucleic acid complex (seeUS Patent Application Publication 20070218122). In further embodiments,the carrier system is a protein-based carrier system such as a cationicpeptide-nucleic acid complex. A RNAi molecule may also be delivered asnaked siRNA.

Pharmaceutical Compositions:

The compounds disclosed herein may be included in pharmaceuticalcompositions (including therapeutic and prophylactic formulations),typically combined together with one or more pharmaceutically acceptablevehicles or carriers and, optionally, other therapeutic ingredients (forexample, antibodies and/or anti-inflammatory compounds). Thecompositions disclosed herein may be advantageously combined and/or usedin combination with other agents used in order to treat psoriasis.

Such pharmaceutical compositions can be administered to subjects by avariety of mucosa administration modes, including by oral, rectal,intranasal, intrapulmonary, transdermal, or by topical delivery to theskin.

To formulate the pharmaceutical compositions, the compound can becombined with various pharmaceutically acceptable additives, as well asa base or vehicle for dispersion of the compound. Desired additivesinclude, but are not limited to, pH control agents, such as arginine,sodium hydroxide, glycine, hydrochloric acid, citric acid, and the like.In addition, local anesthetics (for example, benzyl alcohol),isotonizing agents (for example, sodium chloride, mannitol, sorbitol),adsorption inhibitors (for example, Tween 80), solubility enhancingagents (for example, cyclodextrins and derivatives thereof), stabilizers(for example, serum albumin), and reducing agents (for example,glutathione) can be included.

When the composition is a liquid, the tonicity of the formulation, asmeasured with reference to the tonicity of 0.9% (w/v) physiologicalsaline solution taken as unity, is typically adjusted to a value atwhich no substantial, irreversible tissue damage will be induced at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about 0.3 to about 3.0, such as about 0.5 toabout 2.0, or about 0.8 to about 1.7.

The compound can be dispersed in a base or vehicle, which can include ahydrophilic compound having a capacity to disperse the compound, and anydesired additives. The base can be selected from a wide range ofsuitable compounds, including but not limited to, copolymers ofpolycarboxylic acids or salts thereof, carboxylic anhydrides (forexample, maleic anhydride) with other monomers (for example, methyl(meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers,such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone,cellulose derivatives, such as hydroxymethylcellulose,hydroxypropylcellulose and the like, and natural polymers, such aschitosan, collagen, sodium alginate, gelatin, hyaluronic acid, andnontoxic metal salts thereof. Often, a biodegradable polymer is selectedas a base or vehicle, for example, polylactic acid, poly(lacticacid-glycolic acid) copolymer, polyhydroxybutyric acid,poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.Alternatively or additionally, synthetic fatty acid esters such aspolyglycerin fatty acid esters, sucrose fatty acid esters and the likecan be employed as vehicles. Hydrophilic polymers and other vehicles canbe used alone or in combination, and enhanced structural integrity canbe imparted to the vehicle by partial crystallization, ionic bonding,cross-linking and the like. The vehicle can be provided in a variety offorms, including fluid or viscous solutions, gels, pastes, powders,microspheres, and films for direct application to a mucosa I surfacesuch as skin.

The compound can be combined with the base or vehicle according to avariety of methods, and release of the compound can be by diffusion,disintegration of the vehicle, or associated formation of waterchannels. In some circumstances, the compound is dispersed inmicrocapsules (microspheres) or nanoparticles prepared from a suitablepolymer, for example, 5 isobutyl 2-cyanoacrylate (see, for example,Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in abiocompatible dispersing medium, which yields sustained delivery andbiological activity over a protracted time.

The pharmaceutical compositions of the disclosure can alternativelycontain as pharmaceutically acceptable vehicles substances as requiredto approximate physiological conditions, such as pH adjusting andbuffering agents, tonicity adjusting agents, wetting agents and thelike, for example, sodium acetate, sodium lactate, sodium chloride,potassium chloride, calcium chloride, sorbitan monolaurate, andtriethanolamine oleate. For solid compositions, conventional nontoxicpharmaceutically acceptable vehicles can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like. Pharmaceutical compositions foradministering the compound can also be formulated as a solution,microemulsion, or other ordered structure suitable for highconcentration of active ingredients. The vehicle can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol, and thelike), and suitable mixtures thereof. Proper fluidity for solutions canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of a desired particle size in the case of dispersibleformulations, and by the use of surfactants. In many cases, it will bedesirable to include isotonic agents, for example, sugars, polyalcohols,such as mannitol and sorbitol, or sodium chloride in the composition.Prolonged absorption of the compound can be brought about by includingin the composition an agent which delays absorption, for example,monostearate salts and gelatin.

In certain embodiments, the compound can be administered in a timerelease formulation, for example in a composition which includes a slowrelease polymer. These compositions can be prepared with vehicles thatwill protect against rapid release, for example a controlled releasevehicle such as a polymer, microencapsulated delivery system orbioadhesive gel. Prolonged delivery in various compositions of thedisclosure ca n be brought about by including in the composition agentsthat delay absorption, for example, aluminum monostearate hydrogels andgelatin. When controlled release formulations are desired, controlledrelease binders suitable for use in accordance with the disclosureinclude any biocompatible controlled release material which is inert tothe active agent and which is capable of incorporating the compoundand/or other biologically active agent. Numerous such materials areknown in the art. Useful controlled-release binders are materials thatare metabolized slowly under physiological conditions following theirdelivery (for example, at a mucosa I surface, or in the presence ofbodily fluids). Appropriate binders include, but are not limited to,biocompatible polymers and copolymers well known in the art for use insustained release formulations. Such biocompatible compounds arenon-toxic and inert to surrounding tissues, and do not triggersignificant adverse side effects, such as nasal irritation, immuneresponse, inflammation, or the like. They are metabolized into metabolicproducts that are also biocompatible and easily eliminated from thebody.

Exemplary polymeric materials for use in the present disclosure include,but are not limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolyzable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids and polylactic acids, poly(DL-lacticacidco-glycolic acid), poly(D-lactic acid-co-glycolic acid), andpoly(L-lactic acid-coglycolic acid). Other useful biodegradable orbioerodable polymers include, but are not limited to, such polymers aspoly(epsilon-caprolactone), poly(epsilon-aprolactone-CO-lactic acid),poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyricacid), poly(alkyl-2-cyanoacrilate), hydrogels, such as poly(hydroxyethylmethacrylate), polyamides, poly(amino acids) (for example, L-leucine,glutamic acid, L-aspartic acid and the like), poly(ester urea),poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,polyorthoesters, polycarbonate, polymaleamides, polysaccharides, andcopolymers thereof. Many methods for preparing such formulations arewell known to those skilled in the art (see, for example, Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978). Other useful formulations includecontrolled-release microcapsules (U.S. Pat. Nos. 4,652,441 and4,917,893), lactic acid-glycolic acid copolymers useful in makingmicrocapsules and other formulations (U.S. Pat. Nos. 4,677,191 and4,728,721) and sustained-release compositions for water solublepeptides.

The pharmaceutical compositions of the disclosure typically are sterileand stable under conditions of manufacture, storage and use. Sterilesolutions can be prepared by incorporating the compound in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecompound and/or other biologically active agent into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated herein. In the case of sterilepowders, methods of preparation include vacuum drying and freeze-dryingwhich yields a powder of the compound plus any additional desiredingredient from a previously sterile-filtered solution thereof. Theprevention of the action of microorganisms can be accomplished byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

RNAi/Aptamer Chimeras

Aptamers are single stranded oligonucleotides selected from randomsequence libraries with high affinity and specificity to the targetmolecules (Zhou J et al, Mol Ther 21, 192-200 (2013); Ni X et al, CurrMed Chem 18, 4206-4214 (2011); both of which are incorporated byreference herein). Besides being effective therapeutic agents, aptamershave been actively exploited for targeted delivery of drugs includingRNAi Zhou J et al, Front Genet 3, 234 (2012); incorporated by referenceherein). In theory, due to their high specificity and affinity, aptamerscan deliver RNAi into any cell type provided the cells express theligand for aptamer to bind. Aptamer RNAi chimeras, first described inMcNamara J O et al, Nat Biotechnol 24, 1005-1015 (2006) (which isincorporated by reference herein) has been used to deliver RNAi intoprostate cancer cells. An aptamer-RNAi chimera with an aptamer thatbinds specifically to HIV envelope protein and expressed by viralinfected T cells and RNAi to viral genes and successfully suppressed HIVreplication in HIV infected human CD4+ T cells (Zhou J et al, Mol Ther16, 1481-1489 (2008); incorporated by reference herein). A CD4aptamer-RNAi chimera targeting CCR5, gag and vif was delivered toinfected human CD4+ T cells, suppressed the expression of the targetedgenes and killed HIV (Wheeler L A et al, J Clin Invest 1212401-2412(2011) and Wheeler L A et al, Mol Ther 21, 1378-1389 (2013); both ofwhich are incorporated by reference herein.

IL-17 and Disease

Disclosed herein is a CD4 aptamer-shRNA chimera specific to RORγt tosuppress T helper 17 (Th17) cells that has the potential to be a Th17specific therapeutic agent in Th17 mediated inflammatory diseases.Increasing evidence indicates that Th17 cells and their releasedcytokines play a critical role in the pathogenesis of autoimmune andinflammatory diseases (Miossec P et al, Nat Rev Drug Discov 11, 763-776(2012); incorporated by reference herein). Th17 cells preferentiallyexpress and produce IL-17A, IL-17F, IL-21, and IL-22. Th17 cells andtheir secreted cytokines are considered to account for initiation andmaintenance of several autoimmune and inflammatory disorders (vanHamburg J P et al, Ann Rheum Dis 72, 1700-1707 (2013); van Hamburg J Pet al, Arthritis Rheum 63, 73-83 (2011); Cascao R et al, Arth Res Ther12, R196 (2010); all of which are incorporated by reference herein.Blocking IL-17A activity has been proven to be highly effective to treatimmune mediated inflammatory disease models and clinical trials withblocking IL-17 are ongoing with promising results to treat inflammatorydiseases (Chu C Q et al, Ann Rheum Dis 62, 983-990 (2003); Lubberts E,Cytokine 41, 84-91 (2008); and Genovese M C et al, Arthritis Rheum 62,929-939 (2010); all of which are incorporated by reference herein.However, IL-17A and IL-17F are also produced by many other innate immunecells and are important cytokines in host defense (Korn T et al, Ann RevImmuno/27, 485-517 (2009); incorporated by reference herein). Moreover,it is Th17 cells that are detrimental and are to be blocked fortherapeutic purposes. Therefore, it is highly desirable to narrow thetarget to Th17 cells and spare IL-17 cytokines produced by innate immunecells from being blocked.

Psoriasis and IL-17

Psoriasis is common inflammatory skin disease affecting up to 2% of thepopulation (Nestle F O et al, N Engl J Med 361, 496-509 (2009);incorporated by reference herein) with significant co-morbidities(Gelfand J M et al, J Am Med Assoc 296, 1735-1741 (2006) and Neimann A Let al, J Am Acad Derm 55, 829-835 (2006); both of which are incorporatedby reference herein). Psoriasis can have profound psychological effectswith quality-of-life impairment to a similar or worse extent to thatseen in chronic diseases such as cancer, arthritis and depression,resulting in decreased productivity and social functioning (Feldman S Ret al, J Am Acad Derm 37, 564-569 (1997); incorporated by referenceherein). While psoriasis patients with severe disease require systemictherapy the majority (75%) of patients has mild to moderate disease thatis better managed with topical agents and/or phototherapy.

The Th17 subset of helper T cells play a critical role in thepathogenesis of psoriasis. This has been demonstrated in clinical trialsof the monoclonal antibody ustekinumab, which targets the common p40subunit of IL-23 and IL-12. Ustekinumab shows high efficacy in psoriasisand has been approved by FDA for treating psoriasis. In addition,several monoclonal antibodies that specifically target the common p19subunit of IL-23, IL-17A, and the IL-17 receptor are in various stagesof clinical trials for treating psoriasis (reviewed in Garber K, NatBiotechnol 30, 475-477 (2012); incorporated by reference herein.

IL-17 and other members of IL-17 family are produced by many other celltypes, in particular, innate immune cells which play an important rolein host defense. For example, IL-17 secreting y/8 T cells are criticalfor skin defense to infections (Gray E E et al, J Immunol 185, 6091-6095(2011) and Sumaria N et al, J Exp Med 208, 505-518 (2011); both of whichare incorporated by reference herein.) Furthermore, studies using geneknockout mice demonstrate that y/8 T cells secreting IL-17, but notIL-22 or IL-21, nor α/β T cells are essential for skin defense toStaphylococcus aureus infection. IL-17 secreting γ/δ T cells are presentin psoriatic lesions but their role in the pathogenesis of psoriasis hasbeen questioned (Cai Y et al, Immunity 35, 596-610 (2011); incorporatedby reference herein). Systemic blockade of IL-17 potentially results inan increased risk of infection.

Because γ/δ T cells do not express CD4, the disclosed composition willselectively target on Th17 cells and spare γ/δ T cells. Therefore anon-systemic approach to the treatment of psoriasis that selectivelytargets Th17 cells will be highly advantageous.

Aptamer-RNAi Chimeras to Target Rory

Rory is a nuclear transcription factor. The intracellular location ofRory makes it an undruggable target by monoclonal antibodies. It wasrecently reported that digoxin and its derivatives can suppress Th17cells by antagonizing Rory (Huh J R et al, Nature 472, 486-490 (2011)and Fujita-Sato S et al, J Biol Chem 286, 31409-31417 (2011); both ofwhich are incorporated by reference herein). However, the narrowtherapeutic index of digoxin and its effects on cardiac rhythm (Bhatia SJ and Smith T W, J Cardiac Surg 2, 453-465 (1987); incorporated byreference herein) are likely to limit its use for therapy in psoriasis.An approach using RNAi to directly target RORγt expression would be morespecific with the potential for fewer side effects. An RNAi specific toRORγt can be delivered intracellularly, and would be easilymanufactured.

RNAi based therapeutics have been evaluated in several preclinical tophase II clinical trials for diseases ranging from asthma and cancer toviral infections (Vaishnaw A K et al, Silence 1, 14 (2010), incorporatedby reference herein. However, the major hurdle for widespread use ofRNAi as therapeutic agents is the inefficient intracellular RNAidelivery to target sites.

Aptamers are single-stranded nucleic acids that bind to moleculartargets with high affinity and specificity due to their stable threedimensional shapes (Bouchard P R et al, Ann Rev Pharm Tax 50, 237-257(2010); incorporated by reference herein). Aptamers are typically 20-100nucleotides in length and ca n be selected from libraries of up to 1015individual sequences to bind with high affinity to a wide array ofproteins and/or to modulate protein function in a manner analogous toantibodies (Keefe A D et al, Nat Rev Drug Discovery 9, 537-550 (2010);incorporated by reference herein). Aptamers more readily allow chemicalsubstitutions and other modifications and aptamers elicit minimalimmunogenicity relative to antibodies. Their relatively small physicalsize allows better tissue penetration than antibodies. Aptamers allowfor a straightforward chemical synthesis, which in turn allows easiermodification and rapid in vitro selection. Pegaptanib, an RNA aptamerspecific for VEGF, has been approved by FDA to treat wet age-relatedmacular degeneration (Campa C and Harding S P, Curr Drug Targets 12,173-181 (2011); incorporated by reference herein). Other aptamers arebeing explored for therapeutic purposes.

Use of aptamers as drug delivery devices has been investigatedextensively. Aptamer-RNAi chimeras have been described in, for exampleMcNamara J O et al, Nature Biotechnology 24, 1005-1015 (2006), which isincorporated by reference herein. In that example, a prostate specificmembrane antigen aptamer was conjugated to a polo-like kinase-1 RNAi andused to suppress tumor growth. To date, a variety of aptamer-RNAichimeras have been developed and tested in pre-clinical models fortherapy in viral infections and tumors. For instance, a human CD4aptamer-RNAi to CCR5 chimera can be used to inhibit HIV replication inHIV infected CD4+ T cells in vivo in humanized mice (Wheeler L A et al,J Clin Invest 121, 2401-2412 (2011); incorporated by reference herein.

A chimera comprising a gp120 specific aptamer and a tat/rev RNAi hasbeen constructed. When injected systemically using a Hu-scid mouse modelof HIV infection, this composition inhibits HIV replication and preventsCD4+ T cells from being depleted. Neff C P et al, Science Transl Med 3,66ra66 (2011); incorporated by reference herein.

Treatment of Psoriasis

The administration of the disclosed compounds and pharmaceuticalcompositions can be for prophylactic or therapeutic purposes. Whenprovided prophylactically, the compound is provided in advance of anysymptom. The prophylactic administration of the compound serves toprevent or ameliorate any subsequent disease process. When providedtherapeutically, the compound is provided at or after the onset of asymptom of psoriasis.

For prophylactic and therapeutic purposes, the compound ca n beadministered to the subject topically or via another mucosal deliveryover an extended time period, or in a repeated administration protocol(for example, by an hourly, daily or weekly, repeated administrationprotocol). The therapeutically effective dosage of the compound ca n beprovided as repeated doses within a prolonged prophylaxis or treatmentregimen that will yield clinically significant results to alleviate oneor more symptoms or detectable conditions associated with a targeteddisease or condition as set forth herein. Determination of effectivedosages in this context is typically based on animal model studiesfollowed up by human clinical trials and is guided by administrationprotocols that significantly reduce the occurrence or severity oftargeted disease symptoms or conditions in the subject. Suitable modelsin this regard include, for example, murine, rat, avian, porcine,feline, non-human primate, and other accepted animal model subjectsknown in the art. Alternatively, effective dosages can be determinedusing in vitro models (for example, immunologic and histopathologicassays).

Using such models, only ordinary calculations and adjustments arerequired to determine an appropriate concentration and dose toadminister a therapeutically effective amount of the compound (forexample, amounts that are effective to elicit a desired immune responseor alleviate one or more symptoms of a targeted disease). In alternativeembodiments, an effective amount or effective dose of the compound maysimply inhibit or enhance one or more selected biological activitiescorrelated with a disease or condition, as set forth herein, for eithertherapeutic or diagnostic purposes.

The actual dosage of the compound will vary according to factors such asthe disease indication and particular status of the subject (forexample, the subject's age, size, fitness, extent of symptoms,susceptibility factors, and the like), time and route of administration,other drugs or treatments being administered concurrently, as well asthe specific pharmacology of the compound for eliciting the desiredactivity or biological response in the subject. Dosage regimens can beadjusted to provide an optimum prophylactic or therapeutic response. Atherapeutically effective amount is also one in which any toxic ordetrimental side effects of the compound and/or other biologicallyactive agent is outweighed in clinical terms by therapeuticallybeneficial effects. A nonlimiting range for a therapeutically effectiveamount of a compound and/or other biologically active agent within themethods and formulations of the disclosure is about 0.01 mg/kg bodyweight to about 100 mg/kg body weight, such as about 0.05 mg/kg to about50 mg/kg body weight, or about 0.5 mg/kg to about 5 mg/kg body weight.Dosage can be varied to maintain a desired concentration at a targetsite (for example, psoriatic skin lesions). Dosage can also be adjustedbased on the release rate of the administered formulation, for example,the release rate of a powder, gel, liquid, cream, lotion, or othertopical formulation.

The instant disclosure also includes kits, packages and multi-containerunits containing the herein described pharmaceutical compositions,active ingredients, and/or devices and consumables that facilitate theadministration the same for use in the prevention and treatment ofdiseases and other conditions in mammalian subjects.

In one example, this component is formulated in a pharmaceuticalpreparation for delivery to a subject. The conjugate is optionallycontained in a bulk dispensing container or unit or multiunit dosageform. Optional dispensing devices can be provided, for example a sprayor tube applicator. Packaging materials optionally include a label orinstruction indicating for what treatment purposes and/or in what mannerthe pharmaceutical agent packaged therewith can be used.

Examples Example 1—Synthesis of CD4 Aptamer-RORy shRNA Chimera

Chimera synthesis was modified from previously described methods(Wheeler et al, 2011 supra; Zhou J and Rossi J J, Methods Mol Biol 721,355-371 (2011); Ni X et al, J Clin Invest 121, 2383-2390 (2011); Davis KA, Nuc Acids Res 26, 3915-3924 (1998); all of which are incorporated byreference herein). A cDNA Template with a T7 promoter used for synthesisof the chimera was synthesized with Pfu DNA polymerase (Thermo FisherScientific) and purified with QIAquick® Gel purification kit (Qiagen).The sequence of the cDNA was verified by DNA sequencing. The RNA CD4aptamer-shRNA chimera was transcribed using T7 polymerase in vitro usinga DuraScribe® kit (Illumina). 2′-F-dCTP and 2′-F-dUTP were incorporatedto enhance RNase resistance and Cy3-CTP (GE) was incorporated(Cy3-CTP/2′-F-dCTP ratio=1/9) for visualization. The chimera was phenolextracted and precipitated with sodium acetate/ethanol. Afterresuspension, the chimera was resolved on a 6% dPAGE gel and visualizedusing Cy3 scanning and ethidium bromide staining. It was then purifiedwith G25 column (GE). The sequences of the chimeras of CD4 or mock CD4aptamer-shRNAs against retinoic related orphan receptor (ROR)γ t andCCR5 or scrambled shRNA are shown in the sequence listing above.

In order to investigate if the CD4 aptamer shRNA chimera transcribed invitro is the substrate for the endoribonuclease Dicer that processeslonger endogenous RNA precursors into short RNA as an intracellular stepof the RNAi pathway, Dicer cleavage of the chimera was assayed in vitrowith a recombinant human Dicer kit (Genlantis) according to themanufacturer's instructions.

T lymphocyte cell lines and T-enriched PBMC's Karpas 299 cells(described in Zhang P et al, Lab Invest 89, 1423-1432 (2009);incorporated by reference herein) were maintained in RPM11640 containing10% FBS. For evaluation of Cy3-labeled chimera internalization, Karpas299 cells were incubated with 200 nM chimera overnight. For analysis ofthe function of chimeras in silencing RORγt and IL-17 production, Karpas299 cells were incubated with the chimera for 72 h. Fresh PMBCs fromhealthy donors were isolated by Ficoll (GE) density centrifugation andcultured in RPMI 1640 medium containing 10% human AB serum. T enrichedPBMCs were prepared by adding anti-CD11c, CD11b, CD19, CD56 andimmnunomagnetic beads to PBMCs (BD Bioscience) and purified CD4+ primaryT cells are derived by removing CD8⁺ T cell from T enriched PBMCs withanti-CD8 and immnunomagnetic beads.

Fluorescent Microscopy and Flow Cytometry

Internalization of the synthesized chimera was determined by incubating200 nM or 1 μM Cy3-labeled chimera with Karpas 299 cells or T-cellenriched PBMC overnight. The cells were stained with FITC-anti-CD4(Biolegend) and analyzed by confocal microscopy. T-cell enriched PBMCswere stimulated with anti-CD3/CD28 conjugated to MACS beads for 5 days.

For Th1 cells, IL-12 (10 ng/ml) was added; for Th2 cells, IL-4 (10ng/ml) and anti-human IFN-y (10 μg/ml) were added; for Th17 cells, LPS(100 ng/ml) was added in the culture. PMA (50 ng/ml) and lonomycin (500ng/ml) were added 5 h prior to harvest for intracellular staining.Intracellular staining for RORγt and IL-17A was performed withPE-anti-mouse/human RORγt and PE-anti human IL-17A (eBioscience);staining for IFN-y and IL-4 was performed with PE-anti-human IFN-γ andPE-anti-human IL-4 (Biolegend) and analyzed by flow cytometry.

Real-Time PCR

Real-time PCR was performed as described in Yomogida K et al, Cytokine58, 431-436 (2012); incorporated by reference herein. The probe andprimers mixes for RORC (Hs01076112), TBX21 (Hs00203436), GATA3(Hs00231122) and GUSB (Hs9999908) were purchased from Thermo FisherScientific. MRNA levels for RORC,TBX21 and GATA3 were normalized byGUSB.

Quantification of Cytokines

IL-17A levels in the supernatant were quantified by ELISA (eBiosciences)as described in Yomogida K et al, Cytokine 63, 6-9 (2013); incorporatedby reference herein. Karpas 299 cells were incubated with 50 ng/ml PMAand additional 40 mM sodium chloride for 48 h prior to harvesting thesupernatant. T-cell enriched PBMCs were activated with biotinylatedantibodies against human CD3 and CD28, conjugated to anti-biotin MACSbeads (Miltenyi Biotec Inc.) and 100 ng/ml lipopolysaccharide (LPS) 48 hprior to collecting the supernatant.

Statistics

Data are presented as mean±SD. Data of real-time PCR, ELISA and flowcytometry were analyzed by one-way ANOVA followed by Dunnett comparisontest. P value <0.05 was considered significant.

Example 2—CD4 Aptamer-RORγt Sh RNA Chimera was Specifically Internalizedinto Human CD4⁺ T Cells

An RNA aptamer that was identified by SELEX as able to specifically bindcellular membrane proteins with high-affinity is described in Bouchard PR et al, Ann Rev Pharmacol Toxicol 50, 237-257 (2010); incorporated byreference herein. It is also known that CD4 RNA aptamers can conjugateand deliver siRNAs/shRNAs targeting CCR5 and HIV gp120 gene into the Tcells that express CD4 (Jayasena et al, U.S. Pat. No. 5,869,641 (1999);incorporated by reference herein). Described herein is the generation ofa cDNA template that encodes a CD4 aptamer, a RORγt shRNA sense chain, aloop and a RORγt shRNA antisense chain. The cDNA sequence was verifiedby sequencing. RNA from the construct (CD4-AshR-RORγt) was transcribedas a single molecule in vitro by T7 polymerase transcription.

A mock CD4 aptamer (SEQ ID NO: 2) was created using scrambled sequence.Both CD4-AshR-RORγt and mock-CD4-AshR-RORγt chimeras are 133 nucleotidesin length (FIG. 1A). The predicted secondary structures ofCD4-AshR-RORγt (FIG. 1B) and mock CD4-AshR-RORγt chimeras (FIG. 1C).Similarly, CD4 aptamer-CCR5 shRNA (CD4-AshR-CCR5) and CD4aptamer-scrambled shRNA (CD4-AshR-scrambled) chimeras were generated asnegative controls for RORγt shRNA. All of the chimeras incorporated2′-F-CTP and 2′-F-UTP for enhanced resistance to RNase. In order totrack internalization of the chimeras, two Cy3-CTPs were incorporatedinto each chimera as determined by spectophotometric analysis. A strongfluorochrome signal was readily detected by fluorescent gel scanning(FIG. 2A). Consistent with the characteristic of specifically andeffectively delivering the Cy3-labeled CD4-AshR-RORγt entered human CD4⁺T cell line, Karpas 299 cells and CD4⁺ T cells in PBMC, as assessed withfluorescent confocal microscope and flow cytometric analysis (FIGS. 2B,2C and 2D). In contrast, Cy3-labeled mock CD4-AshR-RORγt, in which thesequence of CD4 aptamer was scrambled, was unable to be internalizedinto Karpas 299, nor in CD4⁺ T cells (FIG. 2D). Only a negligible amountof CD4 aptamer or CD4 aptamer conjugated to siRNAs is up-taken by CD8⁺ Tcells (FIG. 2D). These data suggest that the synthesized CD4aptamer-shRNA chimera can be uniquely and sufficiently transferred intothe CD4⁺ human T cells.

Several strategies have been exploited to link an RNAi to an aptamer(Dassie J P et al, Ther Deliv 4, 1527-1546 (2013); incorporated byreference herein). Aptamer-RNAi chimeras linking an aptamer with an RNAidirectly without using a linker sequence provides effective and specificdelivery of RNAi into target cells. To make an aptamer-RNAi chimera, anaptamer-siRNA-sense strand is transcribed then is annealed to theseparately synthesized antisense of siRNA. It was found that theannealing efficiency of antisense to sense strand linked to the aptameris not consistent. Whereas, aptamer-shRNA chimera has a unique advantagebeing synthesized as a single RNA strand which does not requireannealing with other RNAs. High yield production of aptamer-shRNA as asingle molecule can be consistently achieved. This is particularlyimportant for large scale of production of aptamer-shRNA for in vivouse. Moreover, the RNAi moiety of aptamer-shRNA chimera folds into ashort hairpin structure (FIGS. 1B and C) which closely resemblesendogenous microRNA. This has been demonstrated to be more readilyprocessed by the RNAi machinery.

Example 3—a CD4-AshR-RORγt Chimera Significantly Silences RORγtExpression in Human CD4⁺ T Cells

Intracellular small hairpin RNA is cleaved into 21-25 nucleotide doublestranded RNA by Dicer and then the guide strand of the resultingduplexes are processed to the RNA-induced silencing complex (RISC) todegrade the complementary mRNA. Consistent with this, as shown in theFIGS. 1A and D, the size of CD4-AshR-RORγt chimera produced in vitro byT7 RNA polymerase transcription was originally 133 nucleotides inlength. The sh RNA moiety of CD4-AshR-RORγt chimera was released intoshort paired double stranded RNA after cleavage by Dicer (FIG. 1D). Toconfirm the silencing effect on specific gene expression, the level ofRORγt mRNA was reduced by CD4-AshR-RORγt in a concentration-dependentfashion in the CD4⁺ Karpas 299 cells and T cell-enriched PBMCs, but notby mock CD4-AshR-RORγt, CD4-AshR-scrambled control, or CD4-AshR-CCR5(FIG. 3A-3C), as assayed by quantitative real-time PCR. This was furtherdemonstrated by intracellular RORγt staining with flow cytometry (FIG.3D-F). The suppressive effect of CD4-AshR-RORγt delivered specific shRNAon RORγt expression is consistent with specific siRNAs transfected bylipid transfection agents (Burgler S et al, J Immunol 184, 6161-6169(2010); incorporated by reference herein). In contrast, expression ofTBX21 and GATA3 was not altered by CD4-AshR-RORγt (FIG. 3C). These datademonstrated that CD4-AshR-RORγt specifically suppressed RORγt geneexpression.

Example 4—a CD4-AshR-RORγt Chimera Significantly Inhibits 1117Production by CD4⁺ Human T Cells

Down-regulation of RORγt function by its antagonists like digoxinderivatives could result in decrease of both Th17 cells and IL17production (Huh J R (2011) supra.) As shown in FIG. 4A-D, consistentwith decreased RORγt, CD4-AshR-RORγt exerted a concentration-dependentsuppression of IL-17A production in CD4⁺ Karpas 299 cells and Tcell-enriched PBMC.

In parallel with altered secretion of IL-17A, intracellular IL-17Astaining is significantly impaired by CD4-AshR-RORγt, whereas mockCD4-AshR-RORγt, CD4-AshR-scrambled control or CD4-AshR-CCR5 showed noeffect. As shown in the FIG. 4E-H, the intracellular staining for IFN-yand IL-4 was not changed by CD4-AshR-RORγt, suggesting it did not affectthe synthesis of Th1 or Th2 cytokines. This further confirmed that RORγtis a valid target for regulating Th17 cell differentiation and IL-17production.

The results disclosed herein show that a CD4 aptamer can serve as adelivery vehicle for shRNA that targets a specific gene in CD4+ human Tcells. The internalized RORγt shRNA-CD4 aptamer can be cleaved andreleased by Dicer and then specifically silenced the targeted RORγt geneexpression and results in a marked decrease of Th17 differentiation andIL-17 production.

This particular CD4 aptamer does not alter the cell surface levels ofCD4 or other activation markers of the host CD4⁺ T cells. Bysubstituting the shRNA for targeted genes, this CD4 aptamer can be usedas a universal vehicle to introduce RNAi into CD4⁺ T cells. Comparedwith other vehicles for RNAi delivery into T cells, aptamers have manyadvantages. First, the size of aptamers is relatively smaller and lesslikely to be immunogenic. This is particularly critical for in vivo useas therapeutics. Aptamers can be chemically synthesized and it isrelatively less expensive to generate aptamer-shRNA/siRNA. Thus, it isof great interest to evaluate the use of this CD4-AshR-RORγt chimera intreatment of Th17 mediated inflammatory disorders.

Example 5—Testing CD4-AsiC-RORC2 in a Hu-SCIO Psoriasis Model

A model of psoriasis for use in testing topical pharmaceuticalcompositions is described in Chang T et al, Exp Dermatol 20, 555-560(2011) which is incorporated by reference herein. Skin biopsy samples of3-4 cm² would be obtained from psoriatic lesions of patients withpsoriasis and transplanted onto SCID mice at one graft per mouse. Tendays after transplantation, 1×10⁵ Con A-stimulated PBMC obtained fromthe same donor that provided the skin sample would be injectedintradermally into the xenograft. The clinical and histological featuresof psoriasis of the xenograft can be maintained for 16-20 weeks.

Other formulations for topical delivery of CD4-AshR-RORγt chimera caninclude lipid based RNAi delivery media such as GeneCream® provided byTransDerm Inc. This particular composition ca n be used topically totreat arthritis in a mouse model (Takanashi M et al, Gene Therapy 16,982-989 (2009); incorporated by reference herein). Still otherformulations may include DMSO. The formulation used to deliverdiclofenac comprises DMSO, propylene glycol, alcohol, glycerin andpurified water.

Fifteen and 30 μg doses of CD4-AshR-RORγt formulated for topicaladministration would be applied to a psoriatic xenograft once every 3days for a total of 15 days. The xenograft would be harvested forhistological, immunohistochemical and immunological analyses.

For histological analysis, samples would be fixed with formalin andembedded in paraffin for sections and histological staining. Thicknessof the dermis and dermis can be measured. For immunohistochemicalanalysis, samples would be snap frozen and cryosections prepared.Staining with anti-IL-17 and anti-CD4 can be performed to identify Th17cells. Portions of some samples can be digested with collagenase toisolate T cells for flow cytometry. Isolated cells can be stimulatedwith PMA/ionomycin and intracellular staining for IL-17, IL-22, IL-17Fand IFN-y in combination with cell surface staining of CD4 ca n beperformed and analyzed using flow cytometry. Total RNA can be extractedfrom part of the samples and RT-PCR conducted to quantify geneexpression of RORγt and T-bet.

1. A recombinant polyribonucleotide comprising a nucleic acid sequenceof SEQ ID NO:
 1. 2. The recombinant polyribonucleotide of claim 1wherein the polyribonucleotide is chemically synthesized.
 3. Therecombinant polyribonucleotide of claim 1 wherein the polyribonucleotideis transcribed from a deoxyribonucleic acid template.
 4. The recombinantpolyribonucleotide of claim 3 wherein the nucleic acid molecule istranscribed in vitro.
 5. The recombinant polyribonucleotide of claim 1comprising at least one 2′-fluoro ribonucleic acid.
 6. The recombinantpolyribonucleotide of claim 5 comprising 2′-fluoro cytosine and/or2′-fluoro uracil.
 7. An expression vector comprising: a first nucleicacid sequence that encodes SEQ ID NO: 1; and a second nucleic acidsequence comprising a promoter operably linked to the first nucleic acidsequence.
 8. The expression vector of claim 7 stably transfected into acell.
 9. A pharmaceutical composition comprising an effective amount ofthe recombinant polyribonucleotide of claim
 1. 10. The pharmaceuticalcomposition of claim 9 formulated for topical administration.
 11. Amethod of treating a disease mediated by Th17 cells in a subject, themethod comprising: administering the pharmaceutical composition of claim9 to the subject, thereby treating the disease mediated by the Th17cells.
 12. The method of claim 11 wherein the disease comprisesrheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus,psoriasis, inflammatory bowel disease, and type 1 diabetes mellitus.